We have studied the mechanisms that permit and regulate latency and reactivation of herpes simplex viruses types 1 (HSV-1) and 2 (HSV-2) and varicella-zoster virus (VZV). We have been determining the level of latent infection with HSV and VZV in human neurons. The HSV latency associated transcripts (LATs) are the only viral RNAs consistently expressed during latency. While earlier work from our section examined latent viral loads and LATs in extracts from whole ganglia, we have recently developed and standardized assays using laser capture microdissection (LCM) in which single cells or groups of cells can be removed from a histologic section and subjected to quantitative PCR. This allows us to determine the quantity of latent HSV-1 or VZV DNA in neurons in human ganglia, the percentage of neurons harboring latent viral, and the quantity of viral DNA in individual neurons. [unreadable] [unreadable] Acyclovir is the most common drug used to treat HSV infections. Acyclovir must first be phosphorylated by the viral thymidine kinase (TK) so that the drug can be activated to inhibit viral DNA replication. Some immunocompromised patients, who are treated with acyclovir for prolonged periods of time, develop HSV mutants with a change in the viral thymidine kinase gene so that the virus is resistant to the antiviral activity of acyclovir. While acyclovir resistant HSV have been studied in animal models, drug resistant virus has never been detected in latently infected human ganglia.[unreadable] [unreadable] We examined trigeminal and dorsal root ganglia from an immunocompromised woman who died with a lymphoma. She had a history of a chronic HSV-1 infection of the skin and mucous membranes that had been resistant to acyclovir therapy. We detected mutated sequences in the HSV-1 thymidine kinase gene in her ganglia. These thymidine kinase mutations were due to an insertion or deletion of guanines in a region of the protein which normally has 7 consecutive guanines. From 4% to 70% of the HSV-1 thymidine kinase genes were mutated in each ganglion. Most (73%) ganglia had more than one type of mutation in the HSV-1 thymidine kinase genes. When individual neurons were examined, 42% were coinfected with HSV-1 thymidine kinase mutant and wild-type virus without mutations, 29% were infected with wild-type HSV-1 alone, and 29% were infected with only HSV-1 thymidine kinase mutant virus. Thus, HSV-1 with mutations in the thymidine kinase gene can establish latency in human neurons in either the absence or the presence of wild-type virus.